1. Field of the Invention
This invention generally relates to methods for altering one or more parameters of a measurement system. Certain embodiments relate to methods and systems for optimizing one or more parameters of a measurement system for classifying particles of a population.
2. Description of the Related Art
The following descriptions and examples are not admitted to be prior art by virtue of their inclusion within this section.
Generally, flow cytometers provide measurements of fluorescence intensity of laser excited polystyrene beads as they pass linearly through a flow chamber. In some systems, there are four measurements that are performed: the level of light scattered by a bead at 90 degrees to the excitation source, two measurements of fluorescence used to determine the bead “identity” or “classification,” and a third fluorescence measurement typically used to detect and/or quantify a surface chemical reaction of interest. Each of the three fluorescent measurements is made at a different wavelength. These and any other fluorescent measurements are performed by different “channels” of the system (e.g., reporter channels, classification channels), which include a detector and possibly other components (e.g., optical components, electronic components, etc.) coupled to the detector.
In one example, the fluorescence measurement of the chemical reaction is quantified by optically projecting an image of the bead as it passes through the illumination zone of the excitation laser on the photosensitive area of a photomultiplier tube (PMT). The output of the PMT is a current pulse, which is then conditioned by analog electronics and digitized by an analog to digital (A/D) converter. The resultant digital values obtained from the A/D converter may be further conditioned in the digital domain by a digital signal processing (DSP) algorithm. The end product per bead is a single integer value, which is generally proportional to the chemical reaction on the surface of the bead.
Each flow cytometer based system such as the Luminex 100 system, which is manufactured by Luminex Corp., Austin, Tex., displays results of particle measurements (e.g., the 100-region LabMAP) in a slightly different way than a “typical” flow cytometer instrument. These differences in the displays are a result of the accumulated tolerance for many of the components of the system such as the diode laser, the photodiodes, the optical filters, and the electronics used to process the data. In particular, particles are classified by comparing values generated during analysis of the particles to regions located in a classification space (e.g., the Map). Particles having values that are located within a region in the classification space are assigned the classification corresponding to this region. Therefore, in order to account for the accumulated tolerance of the system described above, the size of the regions in the classification space that are used to classify different populations are made larger than necessary to contain the values of the different populations.
One result of using these larger than necessary classification regions is inconsistent classification of particle populations between one system and another. For instance, one system might be able to classify 95% of a particle population as belonging to a particular region, and 0.5% of that population would typically be misclassified in another region, whereas a different system might correctly classify 98% of this particle population and misclassify a smaller percentage of the population. Therefore, using a larger than necessary classification region results in poor system-to-system matching. System-to-system matching, however, may be desirable, for example, when multiple measurement systems are used in a single facility or organization to perform assays on biological samples. In this manner, results obtained using one measurement system may be directly compared to results obtained using a different measurement system.
Obviously, one way to reduce the size of the classification regions is to reduce the accumulated tolerance of the system. One way to reduce the accumulated tolerance of the system is to manufacture the system using components that have extremely narrow tolerances. However, using such components places a significant burden on manufacturing personnel in sourcing these components. In addition, rigorous assembly efforts can be used to try to compensate for tight tolerances. However, like the extremely narrow tolerance components, using rigorous assembly efforts increases the complexity and difficulty of manufacturing. Therefore, currently available methods for reducing the size of the classification regions increase system manufacturing time, decrease manufacturing throughput, and increase overall system cost.
Accordingly, it would be desirable to reduce the size of the classification regions such that the system can classify particles with greater system accuracy and greater system-to-system uniformity without complicating the manufacturing process, increasing the manufacturing time, decreasing manufacturing throughput, and increasing overall system cost.